Expression And Characterization Of Recombinant Flagellin FlgE Of Pseudomonas Aeruginosa

Objective: To construct the prokaryotic expression and purification protocols forPseudomonas aeruginosa type E flagellin (FlgE) and to study its bioactivity.Methods: With analysis of Pseudomonas aeruginosa flagellin FlgE sequences, thewhole-length FlgE gene was amplified from Pseudomonas aeruginosa genomic DNA byusing PCR and primers with proper restriction enzyme sites. The amplified FlgE fragmentand prokaryotic expression plasmid pET24a were digested with NdeI and HindIIIrespectively. The target fragment and vector were recovered and ligated to obtain therecombinant plasmid pET24a-FlgE. DNA sequencing of positive clone confirmed that thetarget gene and the junctions with vectors were all correct. The plasmid pET24a-FlgE wastransformed into BL21bacteria. The culture conditions like temperature, rotation speed,inducer concentrations, time length were optimized to achieve maximal expression of thetarget recombinant FlgE with6xHis tag at C terminal. FlgE-His proteins were purifiedusing His-Trap affinity chromatography columns and identified by SDS-PAGE. Thepurified proteins were further subjected to endotoxin elimination with proper kits. Thepurified recombinant FlgE was added to cultured corneal epithelial cells for4hours andthe expression of several inflammation-related molecules was examined by using real-timequantitative PCR.Results: The recombinant plasmid pET24a-FlgE was successfully constructed and highlevel FlgE expression was achieved in BL21with rotation at16℃and1mmol/Lisopropyl-β-D-glucopyranosyl-galactosidase induction for20hours. Purified recombinantFlgE-His was obtained and used for primary bioactivity assay. After treatment of cornealepithelial cells with20μ g/mL FlgE for4hours, the expression of inflammatory cytokines IL-6, IL-8were significantly increased. Inactivation of the FlgE with ethanol abolished itsstimulatory activity.Conclusion: The prokaryotic expression and purification system for recombinantPseudomonas aeruginosa flagellin FlgE was set up, and the recombinant FlgE stimulatedthe expression of inflammatory factors in corneal epithelial cells.